Summary. In order to investigate the involvement of apoptosis in the pathogenesis of aplastic anaemia (AA) we determined the proportion of apoptotic cells in ...
British Journal of Haematology, 1997, 98, 18–20
SHORT REPORT
Increased apoptotic cells in bone marrow biopsies from patients with aplastic anaemia F E RNA ND O C AL LERA A ND R O B E RTO P. FA L CA˜ O Department of Clinical Medicine, School of Medicine, Ribeira˜o Preto, Sa˜o Paulo, Brazil Received 14 November 1996; accepted for publication 2 April 1997
Summary. In order to investigate the involvement of apoptosis in the pathogenesis of aplastic anaemia (AA) we determined the proportion of apoptotic cells in paraffinembedded bone marrow biopsies from patients with aplastic anaemia using an in situ TdT-catalysed DNA nick end labelling (TUNEL) staining method. A significant increase in the proportion of mononuclear apoptotic cells was demonstrated in biopsies from patients with aplastic anaemia
(8.19 6 1.45%) when compared with controls (2.07 6 0.86%). These data support the view that apoptosis may play a role in the pathophysiology of bone marrow failure.
Apoptosis is a morphologically distinct form of programmed cell death that plays a critical role in the homeostasis of haemopoietic stem cells controlling the rate of production of committed cells through the sensitivity of progenitor cells to the presence or absence of survival factors (Kerr et al, 1972; Williams et al, 1990). Recent evidence suggests that alterations in apoptosis contribute to the pathogenesis of human diseases including cancer, viral infections and autoimmune diseases (Thompson, 1995). Furthermore, other studies have supported the view that apoptosis is involved in the pathophysiology of bone marrow failure in aplastic anaemia (AA). Maciejewski et al (1995) have shown that Fas-receptor (Itoh et al, 1991) expression is increased in bone marrow CD34þ cells of patients with AA. In addition, we have demonstrated that Fas receptor is also overexpressed in bone marrow and peripheral blood lymphocytes of patients with aplastic anaemia (Callera et al, 1995). Finally, Philpott et al (1995) have shown that a significantly larger proportion of CD34þ progenitor cells from aplastic anaemia bone marrow were apoptotic as compared with normal CD34þ bone marrow when estimated by flow cytometry; the most marked difference was observed in the more severely affected AA patients. However, to our knowledge, there are no data concerning the in situ detection of apoptotic cells in bone marrow biopsies of patients with aplastic anaemia. Therefore we investigated the proportion
of mononuclear apoptotic cells in AA marrow biopsies by using an in situ TdT-catalysed DNA nick end labelling (TUNEL) staining method (Gavrieli et al, 1992).
Correspondence: Dr Roberto Passetto Falca˜o, Department of Clinical Medicine, School of Medicine, Ribeira˜o Preto, Sa˜o Paulo, Av. Bandeirantes 3900, 14049-900, Brazil.
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Keywords: aplastic anaemia, apoptosis, bone marrow biopsy, stem cells, TUNEL method.
MATERIAL AND METHODS Posterior iliac crest bone marrow biopsies were obtained from 11 patients with AA aged 16–65 years (five females and six males). Four had severe and seven moderate aplastic anaemia (Camitta et al, 1976). The values (median, range) of haemoglobin, neutrophil and platelet counts were 5.8 g/dl (1.9–10.9), 0.8 × 109/l (1.4–2.4) and 9 × 109 /l (2–20) respectively. All patients were transfusion dependent. Five patients had not been previously treated and six were patients who had relapsed after immunosuppressive therapy or who did not respond to therapy. Marrow biopsies without tumour involvement or histological alterations obtained from 12 patients with stage I Hodgkin disease were studied as controls. Bouin-fixed bone marrow biopsies were embedded in paraffin and 4–6 mm paraffin sections were adhered to slides followed by decalcification. Apoptotic cells were assessed in the deparaffinized and rehydrated slides using in situ terminal deoxynucleotidyl transferasecatalysed DNA nick end labelling (TUNEL) staining (ApopTagTM plus peroxidase kit, ONCOR Inc., U.S.A.). The percentage of positive cells (peroxidase-stained nuclei) was determined by counting 500 mononuclear cells from each slide. q 1997 Blackwell Science Ltd
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Fig 1. Control (A) and aplastic anaemia (B) bone marrow paraffin sections stained by the TUNEL method. Characteristic apoptotic cells are marked by arrows.
RESULTS AND DISCUSSION The proportion of mononuclear apoptotic cells was significantly increased in the AA bone marrow biopsies (8.19 6 1.45%, mean 6 SEM; Mann-Whitney U test, P < 0.05) compared to controls (2.07 6 0.86%) (Fig 2). The labelling target of the TUNEL method is the multitude of new 30 -OH DNA ends generated by DNA fragmentation and typically localized in morphologically identifiable nuclei. Nucleotide residues are catalytically added to the DNA by terminal q 1997 Blackwell Science Ltd, British Journal of Haematology 98: 18–20
deoxynucleotidyl transferase (TdT), an enzyme which catalyses a template-independent addition of nucleotide triphosphate to the 30 -OH ends of double- or single-stranded DNA, permitting the visualization of apoptosis at the single cell level. In contrast, normal or proliferative cells, which have relatively insignificant numbers of DNA 30 -OH ends, do not stain with TUNEL (Gavrieli et al, 1992). The use of this method in bone marrow biopsies of AA enabled the determination of the actual fraction of cells undergoing apoptosis without loss of the histologic structure of the tissue
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Short Report the in situ method since the mononuclear cells obtained by Ficoll-Hypaque gradient separation for cell suspension studies may lead to a selective loss of some cell subsets. The mechanisms underlying increased apoptosis in AA are poorly understood. The recently reported involvement of a Fas-based mechanism of apoptosis in cells from AA patients (Maciejewski et al, 1995; Callera et al, 1995) has suggested that apoptosis may be caused by excessive activity of the Fas system. Understanding of the induction mechanism of Fas expression, the promoter elements of the Fas gene and the intracellular pathway induced by Fas may help to delineate their possible role in controlling the increased apoptosis of AA bone marrow cells. ACKNOWLEDGMENTS
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Fig 2. Percent apoptosis of mononuclear cells from control ( ) and ( ) aplastic anaemia (AA) bone marrow biopsies detected by the TUNEL staining method. Horizontal bars represent the mean values.
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but it did not allow the identification of which specific cell population was stained for apoptosis. Since the marrow biopsies of AA are poor in myeloid cells and contain scattered stromal cells, lymphocytes, macrophages and plasma cells, mature polymorphonuclear cells were not considered for comparison with control slides and only mononuclear cells were counted. Clustered or isolated mononuclear apoptotic cells stained by the TUNEL method were clearly identified in different regions of the AA bone marrow paraffin sections (Fig 1), suggesting that apoptosis is a diffuse phenomenon within the bone marrow of AA patients. These data do not agree with the study of Philpott et al (1995) in which the apoptosis of AA bone marrow mononuclear cells estimated by 7-amino-actinomycin D (7-AAD) staining did not reveal any significant difference between normal and AA samples, although an increased number of apoptotic cells was restricted to the CD34þ bone marrow cells. These contrasting findings could be explained by the characteristics of each method. Although the use of both methods have been employed for verification of apoptosis, TUNEL staining enables in situ identification of very early apoptotic cells preceding the appearance of changes detected by flow cytometry in tissues such as bone marrow (Gavrieli et al, 1992; Schmid et al, 1994). In addition, the possible contamination of bone marrow aspirates with peripheral blood cells can lead to an underestimate of the proportion of apoptotic cells in cell suspensions. Furthermore, the composition of bone marrow aspirates studied by flow cytometry is not the same as that of aspirates analysed by
The authors thank Dr Fernando A. Soares and Aglair B. Garcia. This study was supported by the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and by the Financiadora de Estudos e Projetos (FINEP). REFERENCES Callera, F., Garcia, A.B. & Falca˜o, R.P. (1995) Increased Fas antigen expression on lymphocytes from severe aplastic anemia. Blood, 86, 478a (abstract 1898). Camitta, B.M., Thomas, E.D., Nathan, G.D., Santos, G., GordonSmith, E.C. & Gale, R.P. (1976) Severe aplastic anemia: a prospective study of the effect of early transplantation on acute mortality. Blood, 48, 63–69. Gavrieli, Y., Sherman, Y. & Ben-Sasson, S.A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. Journal of Cell Biology, 119, 93–501. Itoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, S., Sameshima, M., Hase, A., Seto, Y. & Nagata, S. (1991) The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell, 66, 233–243. Kerr, J.F.R., Wyllie, A.H. & Currie, A.R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer, 36, 239–257. Maciejewski, J.P., Selleri, C., Sato, T., Anderson, S. & Young, N.S. (1995) Increased expression of Fas antigen on bone marrow CD34þ cells of patients with aplastic anaemia. British Journal of Haematology, 91, 245–252. Philpott, N.J., Scopes, J., Marsh, J.C.W., Gordon-Smith, E.C. & Gibson, F.M. (1995) Increased apoptosis in aplastic anemia bone marrow progenitor cells: possible pathophysiologic significance. Experimental Hematology, 23, 1642–1648. Schmid, I., Uittenbogaart, C.H., Keld, B. & Giorgi, J.V. (1994) A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry. Journal of Imunological Methods, 170, 145–157. Thompson, C.B. (1995) Apoptosis in the pathogenesis and treatment of disease. Science, 267, 1456–1462. Williams, G.T., Smith, C.A., Spooner, E., Dexter, T.M. & Taylor, D.R., (1990) Haematopoietic colony stimulating factors promote cell survival by suppressing apoptosis. Nature, 343, 76–79.
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